Method of colorimetry and reagent for use therein

ABSTRACT

The present invention provides a colorimetric method that can perform a simple and reliable analysis in a short time. An aqueous solution including a bipyridyl copper complex and glucose dehydrogenase is prepared. A filter paper is impregnated with the aqueous solution and dried. When a sample such as blood is applied to the filter paper, the bipyridyl copper complex produces reddish brown color in accordance with the glucose concentration, and the color produced in the complex is measured. This reaction is a single reaction and thus occurs in a short time (e.g., 5 seconds or less). Since this reaction requires neither hydrogen peroxide nor oxygen, the measured values are highly reliable.

TECHNICAL FIELD

The present invention relates to a colorimetric method and a reagentused for the same.

BACKGROUND ART

In the field of clinical or biochemical examinations, a colorimetricanalysis is employed as a method for analyzing components such asglucose, cholesterol, or the like in a sample. For example, thecalorimetric analysis of glucose is generally as follows: a glucoseoxidase reacts with glucose (substrate) to generate gluconolactone andhydrogen peroxide; and the hydrogen peroxide is detected by a coloringreagent, such as a Trinder's reagent, in the presence of peroxidase.This method, in which the concentration of a substrate is measuredindirectly via hydrogen peroxide, is not limited to glucose, but also isused to analyze other components such as cholesterol.

However, the conventional colorimetric analysis involves the followingproblems. First, the time required for measurement is long because ittakes a long time until a color reaction ends due to the indirectmeasurement of an analyte via hydrogen peroxide. For example, themeasurement of glucose takes 30 to 60 seconds. Second, it is difficultto set conditions because two different enzyme reaction systems shouldbe stabilized simultaneously. Finally, the conventional colorimetricanalysis requires oxygen, and inadequate oxygen supply may lead to aninsufficient reaction.

DISCLOSURE OF INVENTION

With the foregoing in mind, it is an object of the present invention toprovide a calorimetric method that has a single reaction system and canachieve a short-time analysis and provide reliable values with theanalysis.

A colorimetric method of the present invention includes allowing anoxidoreductase to act on an analyte and a color-changeable substancethat changes color by transferring an electron, and performing aqualitative or quantitative analysis of the analyte by measuring colorproduced in the color-changeable substance due to electron transfer fromthe analyte to the color-changeable substance.

This method has a single reaction system, so that the reaction system issimple and stable, and the time until a color reaction ends is short.Therefore, the measuring time is reduced (e.g., when glucose is used asan analyte, the measurement can be performed within about 5 seconds).Moreover, this method requires neither hydrogen peroxide nor oxygen andthus ensures highly reliable values with the analysis.

A reagent of the present invention is used for the above colorimetricmethod and includes an oxidoreductase and a color-changeable substancethat changes color by transferring an electron. A test piece of thepresent invention includes this reagent. Compared with a conventionaltest piece for calorimetric analysis requiring hydrogen peroxide, thetest piece of the present invention can achieve a very short-timeanalysis and ensure highly reliable values with the analysis.

An analysis method that uses a bipyridyl metal complex as alight-emitting substance has been proposed (e.g., JP 10-253633 A).However, this method quite differs from the present invention intechnical field because the present invention relates to a colorimetricmethod. Another technique that allows a bipyridyl metal complex toproduce/erase color by direct application from an electrode has beenproposed (e.g., JP 57-192483 A). However, this technique also quitediffers from the present invention in technical field because thepresent invention utilizes electron transfer by an oxidoreductase.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the dependence of reflectance on glucoseconcentration in an example of the present invention.

FIG. 2 is a graph showing the dependence of reflectance on glucoseconcentration in another example of the present invention.

FIG. 3 is a graph showing a color change in yet another example of thepresent invention.

FIG. 4 is a graph showing a color change in still another example of thepresent invention.

FIG. 5 is a graph showing a color change in still another example of thepresent invention.

FIG. 6 is a graph showing a color change in still another example of thepresent invention.

FIG. 7 is a graph showing the dependence of reflectance on glucoseconcentration in still another example of the present invention.

FIGS. 8A to 8F are graphs showing a color change in still anotherexample of the present invention.

FIGS. 9A to 9D are graphs showing a color change in still anotherexample of the present invention.

FIGS. 10A to 10C are graphs showing a color change in still anotherexample of the present invention.

FIG. 11 is a graph showing a color change in still another example ofthe present invention.

FIG. 12 is a graph showing a color change in still another example ofthe present invention.

FIGS. 13A to 13C are graphs showing a color change in still anotherexample of the present invention.

FIG. 14 is a graph showing a color change in still another example ofthe present invention.

FIG. 15 is a graph showing a color change in still another example ofthe present invention.

FIGS. 16A to 16E are graphs showing a color change in still anotherexample of the present invention.

FIGS. 17A and 17B are graphs showing a color change in still anotherexample of the present invention.

FIGS. 18A to 18C are graphs showing a color change in still anotherexample of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

In a colorimetric method, a reagent, and a test piece of the presentinvention, the color-changeable substance that changes color bytransferring an electron is preferably a transition metal complex. Thetransition metal complex is preferably a copper complex, an ironcomplex, a ruthenium complex, an osmium complex, or a mixture containingat least two of these complexes. It is preferable that a coordinatingatom of a ligand in the transition metal complex is at least oneselected from the group consisting of nitrogen, oxygen, and sulfur. Itis preferable that the ligand is selected, e.g., from the groupconsisting of ammonia, a bipyridyl compound, an imidazole compound, aphenanthroline compound, an ethylenediamine compound, amino acids, atriazine compound, a biquinoline compound, a pyridylazo compound, anitroso compound, an oxine compound, a benzothiazole compound, anacetylacetone compound, an anthraquinone compound, a xanthene compound,oxalic acid, and a derivative of each of the compounds. At least onehydrogen atom that occupies a position other than the coordinationposition of the ligand may be replaced by a substituent. Examples of thesubstituent include an alkyl group, an aryl group, an allyl group, aphenyl group, a hydroxyl group, an alkoxy group, a carboxyl group, acarbonyl group, a sulfone group, a sulfonyl group, a nitro group, anitroso group, a primary amine, a secondary amine, a tertiary amine, anamino group, an acyl group, an amido group, and a halogen group. Thetransition metal complex may include two or more types of ligands, i.e.,it can be a mixed ligand complex.

In a colorimetric method, a reagent, and a test piece of the presentinvention, the oxidoreductase is preferably a dehydrogenase or anoxidase. The reaction rate may increase with the amount of enzyme. Theanalyte is, e.g., glucose, cholesterol, uric acid, lactic acid, pyruvicacid, creatine, or creatinine. In this case, the oxidoreductase may be adehydrogenase or an oxidase that corresponds to the respective analyte.

In a colorimetric method, a reagent, and a test piece of the presentinvention, it is preferable that a mediator is used in addition to thecolor-changeable substance that changes color by transferring anelectron. Although the color-changeable substance can serve as amediator, it has the function not only of transferring an electron butalso changing color. Therefore, the additional mediator differs from thecolor-changeable substance. The mediator increases the speed of electrontransfer, which in turn increases the speed of color change of thecolor-changeable substance. Thus, it is possible to use lessoxidoreductase and obtain a cost advantage. The mediator is, e.g., anosmium complex, a ruthenium complex, or the like. Specific examples ofthe mediator will be described later. When the color-changeablesubstance is used with the mediator, it is preferable that a coppercomplex is used as the color-changeable substance, and an osmium or aruthenium complex is used as the mediator.

A test piece of the present invention preferably includes an inorganicgel as well as the reagent. The color-changeable substance that changescolor by transferring an electron is reduced by electron transfer andthus changes color. However, when oxygen is present in the surroundings,the color may fade due to reoxidation of the color-changeable substance.The inorganic gel can be useful to prevent the fading.

Hereinafter, the present invention will be described in more detail byway of specific examples.

As described above, the color-changeable substance that changes color bytransferring an electron is preferably a transition metal complex in thepresent invention. Among the transition metal complexes particularlysuitable for the color-changeable substance are a copper complex, aniron complex, a ruthenium complex, and an osmium complex.

Copper Complex

The color of the copper complex is changed, e.g., from blue (Cu²⁺) toreddish brown (Cu⁺) by electron transfer that is caused by an enzyme.Examples of a ligand in the copper complex include ammonia, a bipyridylcompound, an imidazole compound, a phenanthroline compound, anethylenediamine compound, amino acids, a triazine compound, abiquinoline compound, a pyridylazo compound, a nitroso compound, anoxine compound, a benzothiazole compound, an acetylacetone compound, ananthraquinone compound, a xanthene compound, oxalic acid, and aderivative of each of the compounds. A mixed ligand with two or moretypes of these ligands may be used.

For the bipyridyl compound, the coordination number is 4 or 6. In viewof stability, two bipyridyls should be coordinated at two coordinationpositions in the complex, respectively. Hydrogen atoms binding to thepyridine rings may be unsubstituted or substituted. The introduction ofa substituent makes it possible to control, e.g., the solubility andoxidation-reduction potential of the complex. Examples of the positionof the substituent include the 4,4′-position and 5,5′-position. Examplesof the substituent include an alkyl group (such as a methyl group, anethyl group, or a propyl group), an aryl group, an allyl group, a phenylgroup, a hydroxyl group, an alkoxy group (such as a methoxy group or anethoxy group), a carboxyl group, a carbonyl group, a sulfone group, asulfonyl group, a nitro group, a nitroso group, a primary amine, asecondary amine, a tertiary amine, an amino group, an acyl group, anamido group, and a halogen group (such as bromine, chlorine, or iodine).

Examples of a bipyridyl copper complex include [Cu(bipyridyl)₂],[Cu(4,4′-dimethyl-2,2′-bipyridyl)₂],[Cu(4,4′-diphenyl-2,2′-bipyridyl)₂], [Cu(4,4′-diamino-2,2′-bipyridyl)₂],[Cu(4,4′-dihydroxy-2,2′-bipyridyl)₂],[Cu(4,4′-dicarboxy-2,2′-bipyridyl)₂],[Cu(4,4′-dibromo-2,2′-bipyridyl)₂], [Cu(5,5′-dimethyl-2,2′-bipyridyl)₂],[Cu(5,5′-diphenyl-2,2′-bipyridyl)₂], [Cu(5,5′-diamino-2,2′-bipyridyl)₂],[Cu(5,5′-dihydroxy-2,2′-bipyridyl)₂],[Cu(5,5′-dicarboxy-2,2′-bipyridyl)₂],[Cu(5,5′-dibromo-2,2′-bipyridyl)₂], [Cu(bipyridyl)₃],[Cu(4,4′-dimethyl-2,2′-bipyridyl)₃],[Cu(4,4′-diphenyl-2,2′-bipyridyl)₃], [Cu(4,4′-diamino-2,2′-bipyridyl)₃],[Cu(4,4′-dihydroxy-2,2′-bipyridyl)₃],[Cu(4,4′-dicarboxy-2,2′-bipyridyl)₃],[Cu(4,4′-dibromo-2,2′-bipyridyl)₃], [Cu(5,5′-dimethyl-2,2′-bipyridyl)₃],[Cu(5,5′-diphenyl-2,2′-bipyridyl)₃], [Cu(5,5′-diamino-2,2′-bipyridyl)₃],[Cu(5,5′-dihydroxy-2,2′-bipyridyl)₃],[Cu(5,5′-dicarboxy-2,2′-bipyridyl)₃], and[Cu(5,5′-dibromo-2,2′-bipyridyl)₃].

For the imidazole compound, the coordination number is 4. Hydrogen atomsbinding to the imidazole rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 2-position, 4-position and5-position. Examples of the substituent include an alkyl group (such asa methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of an imidazole copper complex include [Cu(imidazole)₄],[Cu(4-methyl-imidazole)₄], [Cu(4-phenyl-imidazole)₄],[Cu(4-amino-imidazole)₄], [Cu(4-hydroxy-imidazole)₄],[Cu(4-carboxy-imidazole)₄], and [Cu(4-bromo-imidazole)₄].

The amino acids include, e.g., arginine (L-Arg). A copper complexcontaining arginine generally has the advantage of high solubility. Themixed ligand may be a combination of the bipyridyl compounds and theimidazole compounds or a combination of the bipyridyl compounds and theamino acids, such as [Cu(imidazole)₂(bipyridyl)] or[Cu(L-Arg)₂(bipyridyl)]. The mixed ligand can be used to impart variousproperties to the complex, e.g., arginine improves the solubility of thecomplex.

Iron Complex

The color of the iron complex is changed, e.g., from yellow (Fe³⁺) tored (Fe²⁺) by electron transfer that is caused by an enzyme. Examples ofa ligand in the iron complex include ammonia, a bipyridyl compound, animidazole compound, a phenanthroline compound, an ethylenediaminecompound, amino acids, a triazine compound, a biquinoline compound, apyridylazo compound, a nitroso compound, an oxine compound, abenzothiazole compound, an acetylacetone compound, an anthraquinonecompound, a xanthene compound, oxalic acid, and a derivative of each ofthe compounds. A mixed ligand with two or more types of these ligandsmay be used.

For the bipyridyl compound, the coordination number is 6. Hydrogen atomsbinding to the pyridine rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 4,4′-position and5,5′-position. Examples of the substituent include an alkyl group (suchas a methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of a bipyridyl iron complex include [Fe(bipyridyl)₃],[Fe(4,4′-dimethyl-2,2′-bipyridyl)₃],[Fe(4,4′-diphenyl-2,2′-bipyridyl)₃], [Fe(4,4′-diamino-2,2′-bipyridyl)₃],[Fe(4,4′-dihydroxy-2,2′-bipyridyl)₃],[Fe(4,4′-dicarboxy-2,2′-bipyridyl)₃],[Fe(4,4′-dibromo-2,2′-bipyridyl)₃], [Fe(5,5′-dimethyl-2,2′-bipyridyl)₃],[Fe(5,5′-diphenyl-2,2′-bipyridyl)₃], [Fe(5,5′-diamino-2,2′-bipyridyl)₃],[Fe(5,5′-dihydroxy-2,2′-bipyridyl)₃],[Fe(5,5′-dicarboxy-2,2′-bipyridyl)₃], and[Fe(5,5′-dibromo-2,2′-bipyridyl)3].

For the imidazole compound, the coordination number is 6. Hydrogen atomsbinding to the imidazole rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 2-position, 4-position and5-position. Examples of the substituent include an alkyl group (such asa methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of an imidazole iron complex include [Fe(imidazole)₆],[Fe(4-methyl-imidazole)₆], [Fe(4-phenyl-imidazole)₆],[Fe(4-amino-imidazole)₆], [Fe(4-hydroxy-imidazole)₆],[Fe(4-carboxy-imidazole)₆], and [Fe(4-bromo-imidazole)6].

The amino acids include, e.g., arginine (L-Arg). An iron complexcontaining arginine generally has the advantage of high solubility. Themixed ligand may be a combination of the bipyridyl compounds and theimidazole compounds or a combination of the bipyridyl compounds and theamino acids, such as [Fe(imidazole)₂(bipyridyl)₂] or[Fe(L-Arg)₂(bipyridyl)₂]. The mixed ligand can be used to impart variousproperties to the complex, e.g., arginine improves the solubility of thecomplex.

Ruthenium Complex

Examples of a ligand in the ruthenium complex include ammonia, abipyridyl compound, an imidazole compound, a phenanthroline compound, anethylenediamine compound, amino acids, a triazine compound, abiquinoline compound, a pyridylazo compound, a nitroso compound, anoxine compound, a benzothiazole compound, an acetylacetone compound, ananthraquinone compound, a xanthene compound, oxalic acid, and aderivative of each of the compounds. A mixed ligand with two or moretypes of these ligands may be used.

For the bipyridyl compound, the coordination number is 6. Hydrogen atomsbinding to the pyridine rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 4,4′-position and5,5′-position. Examples of the substituent include an alkyl group (suchas a methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of a bipyridyl ruthenium complex include [Ru(bipyridyl)₃],[Ru(4,4′-dimethyl-2,2′-bipyridyl)₃], [Ru(4,4′-diphenyl-2,2′-bipyridyl)₃], [Ru(4,4′-diamino-2,2′-bipyridyl)₃],[Ru(4,4′-dihydroxy-2,2′-bipyridyl)₃],[Ru(4,4′-dicarboxy-2,2′-bipyridyl)₃],[Ru(4,4′-dibromo-2,2′-bipyridyl)₃], [Ru(5,5′-dimethyl-2,2′-bipyridyl)₃],[Ru(5,5′-diphenyl-2,2′-bipyridyl)₃], [Ru(5,5′-diamino-2,2′-bipyridyl)₃],[Ru(5,5′-dihydroxy-2,2′-bipyridyl)₃],[Ru(5,5′-dicarboxy-2,2′-bipyridyl)₃], and[Ru(5,5′-dibromo-2,2′-bipyridyl)3].

For the imidazole compound, the coordination number is 6. Hydrogen atomsbinding to the imidazole rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 2-position, 4-position and5-position. Examples of the substituent include an alkyl group (such asa methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of an imidazole ruthenium complex include [Ru (midazole)₆],[Ru(4-methyl-imidazole)₆], [Ru(4-phenyl-imidazole)₆][Ru(4-amino-imidazole)₆], [Ru(4-hydroxy-imidazole)₆],[Ru(4-carboxy-imidazole)₆], and [Ru(4-bromo-imidazole)6].

The amino acids include, e.g., arginine (L-Arg). Aruthenium complexcontaining arginine generally has the advantage of high solubility. Themixed ligand may be a combination of the bipyridyl compounds and theimidazole compounds or a combination of the bipyridyl compounds and theamino acids, such as [Ru(imidazole)₂(bipyridyl)₂] or[Ru(L-Arg)₂(bipyridyl)₂]. The mixed ligand can be used to impart variousproperties to the complex, e.g., arginine improves the solubility of thecomplex.

Osmium Complex

The color of the osmium complex is changed, e.g., from orange (Os³⁺) todark brown (Os²⁺) by electron transfer that is caused by an enzyme.Examples of a ligand in the osmium complex include ammonia, a bipyridylcompound, an imidazole compound, a phenanthroline compound, anethylenediamine compound, amino acids, a triazine compound, abiquinoline compound, a pyridylazo compound, a nitroso compound, anoxine compound, a benzothiazole compound, an acetylacetone compound, ananthraquinone compound, a xanthene compound, oxalic acid, and aderivative of each of the compounds. A mixed ligand with two or moretypes of these ligands may be used.

For the bipyridyl compound, the coordination number is 6. Hydrogen atomsbinding to the pyridine rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 4,4′-position and5,5′-position. Examples of the substituent include an alkyl group (suchas a methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of a bipyridyl osmium complex include [Os(bipyridyl)₃],[Os(4,4′-dimethyl-2,2′-bipyridyl)₃],[Os(4,4′-diphenyl-2,2′-bipyridyl)₃], [Os(4,4′-diamino-2,2′-bipyridyl)₃],[Os(4,4′-dihydroxy-2,2′-bipyridyl)₃],[Os(4,4′-dicarboxy-2,2′-bipyridyl)₃],[Os(4,4′-dibromo-2,2′-bipyridyl)₃], [Os(5,5′-dimethyl-2,2′-bipyridyl)₃],[Os(5,5′-diphenyl-2,2′-bipyridyl)₃], [Os(5,5′-diamino-2,2′-bipyridyl)₃],[Os(5,5′-dihydroxy-2,2′-bipyridyl)₃],[Os(5,5′-dicarboxy-2,2′-bipyridyl)₃], and[Os(5,5′-dibromo-2,2′-bipyridyl)3].

For the imidazole compound, the coordination number is 6. Hydrogen atomsbinding to the imidazole rings may be unsubstituted or substituted. Theintroduction of a substituent makes it possible to control, e.g., thesolubility and oxidation-reduction potential of the complex. Examples ofthe position of the substituent include the 2-position, 4-position and5-position. Examples of the substituent include an alkyl group (such asa methyl group, an ethyl group, or a propyl group), an aryl group, anallyl group, a phenyl group, a hydroxyl group, an alkoxy group (such asa methoxy group or an ethoxy group), a carboxyl group, a carbonyl group,a sulfone group, a sulfonyl group, a nitro group, a nitroso group, aprimary amine, a secondary amine, a tertiary amine, an amino group, anacyl group, an amido group, and a halogen group (such as bromine,chlorine, or iodine).

Examples of an imidazole osmium complex include [Os(imidazole)₆],[Os(4-methyl-imidazole)₆], [Os(4-phenyl-imidazole)₆],[Os(4-amino-imidazole)₆], [Os(4-hydroxy-imidazole)₆],[Os(4-carboxy-imidazole)₆], and [Os(4-bromo-imidazole)6].

The amino acids include, e.g., arginine (L-Arg). An osmium complexcontaining arginine generally has the advantage of high solubility. Themixed ligand may be a combination of the bipyridyl compounds and theimidazole compounds or a combination of the bipyridyl compounds and theamino acids, such as [Os(imidazole)₂(bipyridyl)₂] or[Os(L-Arg)₂(bipyridyl)₂]. The mixed ligand can be used to impart variousproperties to the complex, e.g., arginine improves the solubility of thecomplex.

The above explanation of the transition metal complexes is based on thetype of transition metal, and the present invention is not limitedthereto. Hereinafter, the transition metal complexes will be describedbased on their ligands.

A ligand that contains coordinating atoms N, O, and S has groups suchas═N—OH, —COOH, —OH, —SH, and >C═O in the molecule. Examples of metalcomplexes including this type of ligand are NN chelate, NO chelate, NSchelate, OO chelate, OS chelate, SS chelate (bidentate), N chelate(unidentate), and NNN chelate (tridentate). The combination is diverse.When a ligand has a double bond, Cu, Fe, Ru, and Os of the complex tendto have the function of transferring electrons. The ligand preferablyhas an aromatic ring. The ligand may be any of the above substituents.For example, the introduction of a sulfone group can improve thesolubility of the metal complex. The metal complex may be formed bymixing two or more types of ligands and used as a mixed ligand complex.For example, when one of the ligands is amino acids, the metal complexmay have a good affinity with an enzyme. Moreover, various halogen atoms(such as Cl, F, Br, and I) can be attached to part of the site of thecentral metal. The following is an example of the transfer metalcomplexes that are classified by the type of coordination.

NN Coordination Form

Phenanthroline Derivative

-   Cu+1,10-phenanthroline-   Fe+1,10-phenanthroline-   Cu+ bathophenanthroline-   Fe+ bathophenanthroline-   Cu+ bathophenanthroline sulfonic acid-   Fe+ bathophenanthroline sulfonic acid    Bipyridyl Derivative-   Cu+2,2′-bipyridyl-   Fe+2,2′-bipyridyl-   Fe+4,4′-diamino-2,2′-bipyridyl-   Ru+4,4′-diamino-2,2′-bipyridyl    Triazine Derivative-   Cu+ TPTZ (2,4,6-tripyridyl-S-triazine)-   Fe+ TPTZ (2,4,6-tripyridyl-S-triazine)-   Fe+ PDTS (3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-1,2,4-triazine)    Biquinoline Derivative-   Cu+ cuproin (2,2′-biquinoline)    Pyridylazo Derivative-   Fe+ nitro-PAPS    (2-(5-nitro-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl) amino]    phenol)    NO Coordination Form-   Fe+ nitroso-PSAP (2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl) amino]    phenol)-   Fe+ nitroso-ESAP (2-nitroso-5-[N-ethyl-N-(3-sulfopropyl) amino]    phenol)-   Fe+1-nitroso-2-naphthol    NS Coordination Form-   Fe+2-amino-4-thiazole acetic acid    OO Coordination Form-   Fe+1,2-naphthoquinone-4-sulfonic acid    Mixed Ligand Form-   Os+Cl, imidazole, 4,4′-dimethyl-2,2′-bipyridyl-   Os+imidazole, 4,4′-dimethyl-2,2′-bipyridyl-   Cu+ L-arginine, 2,2′-bipyridyl-   Cu+ ethylenediamine, 2,2′-bipyridyl-   Cu+ imidazole, 2,2′-bipyridyl

Next, the colorimetric method of the present invention is applied to atest piece. In this case, a copper complex is used as thecolor-changeable substance that changes color by transferring anelectron, and glucose is used as the analyte. Other analytes such ascholesterol are analyzed basically in the same manner except that theoxidoreductase is changed in accordance with the analytes.

First, a bipyridyl copper complex is prepared either by using acommercially available product or by employing the following manner. Forexample, CuCl₂ and 2,2′-bipyridyl (bpy) are mixed in a water bath atabout 60° C. to 90° C. and synthesized into [Cu(bpy)₂]Cl₂. The molarratio of CuCl₂ to 2,2′-bipyridyl (bpy) is, e.g., 1: 2. The concentrationof the aqueous solution of bipyridyl copper complex ranges, e.g., from 1to 10 wt %. A binder is dissolved in the aqueous solution of bipyridylcopper complex, and then glucose dehydrogenase (GDH) is dissolved in thebinder solution, thus producing a reagent solution. Examples of thebinder include hydroxypropylcellulose (HPC), polyvinyl alcohol (PVA),polyvinyl pyrrolidone (PVP), polyacrylamide, and bovine serum albumin(BSA), and HPC is preferred. The concentration of the binder ranges,e.g., from 0.5 to 5 wt %. The concentration of GDH ranges, e.g., from1000 to 50000 U/ml. A porous sheet (e.g., a filter paper) is impregnatedwith the reagent solution and dried, so that a test piece for glucoseanalysis can be produced. Before impregnation of the reagent solution,it is preferable that the porous sheet is impregnated with an inorganicgel solution and dried. The inorganic gel can be smectite or the like.The concentration of the inorganic gel in the solution ranges, e.g.,from 1 to 5 wt %, preferably 1 to 3 wt %, and more preferably 1.5 to 2wt %. The inorganic gel solution also may include an amphotericsurfactant such as CHAPS. The concentration of the amphoteric surfactantwith respect to the total inorganic gel solution ranges, e.g., from 0.1to 2 wt %, preferably 0.1 to 1 wt %, and more preferably 0.2 to 0.4 wt%. The amount of inorganic gel impregnated into the porous sheet ranges,e.g., from 1 to 50 mg/cm³, preferably 10 to 30 mg/cm³, and morepreferably 15 to 20 mg/cm³, when measured on the basis of the volume ofvoids in the porous sheet. The porous sheet can be an asymmetricalporous film in which pore sizes vary in the thickness direction or inthe sheet surface direction. The original color of this test piece isblue. When a sample containing glucose (e.g., blood) is applied to thetest piece, the color changes to reddish brown in accordance with theglucose concentration. Therefore, the qualitative or quantitativeanalysis of the glucose can be performed using the color change. Thetime required for the analysis is about 2 to 3 seconds after theapplication of the sample. If the test piece is impregnated with aninorganic gel, it is possible to prevent fading caused by reoxidationafter the color change and, e.g., to increase the time from the end of acolor reaction to the start of measuring the color thus produced.

The inorganic gel is preferably selected from swelling clay minerals.Among the swelling clay minerals, bentonite, smectite, vermiculite, orsynthetic fluorine mica is more preferred. In particular, syntheticsmectite such as synthetic hectorite or synthetic saponite, or syntheticmica (the natural mica generally is a non-swelling clay mineral) such asswelling synthetic mica (or Na mica) typified by synthetic fluorine micais preferred.

Next, the colorimetric method of the present invention is applied toliquid system analysis. In this case, a copper complex is used as thecolor-changeable substance that changes color by transferring anelectron, and glucose is used as the analyte. Other analytes such ascholesterol are analyzed basically in the same manner except that theoxidoreductase is changed in accordance with the analytes.

First, a copper complex [Cu(bpy)₂]Cl₂ is synthesized in such a manner asdescribed above. Then, a reagent solution is prepared by dissolving thecopper complex and GDH in a buffer solution. Although they may bedissolved in water, the buffer solution is preferred. The pH of thebuffer solution ranges, e.g., from 6 to 8, and preferably 6.5 to 7. Theconcentration of the copper complex ranges, e.g., from 0.1 to 60 mM,preferably 0.2 to 10 mM, and more preferably 0.3 to 0.6 mM. Theconcentration of GDH ranges, e.g., from 10 to 1000 U/ml, preferably 50to 500 U/ml, and more preferably 100 to 200 U/ml. When a samplecontaining glucose (e.g., blood) is added to the reagent solution, thecolor of the reagent solution changes from blue to reddish brown inaccordance with the glucose concentration in a short time, e.g., 5seconds or less. This change may be observed visually or measured withan optical measuring device such as a spectrophotometer. The amount ofthe added sample ranges, e.g., from 1 to 100 μl, preferably 3 to 10 μl,and more preferably 5 to 10 μl with respect to 1 ml of the reagentsolution.

As described above, it is preferable that a mediator such as an osmiumcomplex or a ruthenium complex is used in addition to thecolor-changeable substance that changes color by transferring anelectron in the colorimetric method of the present invention. For a testpiece, the amount of mediator with respect to the total reagent solutionranges, e.g., from 0.1 to 50 mM, preferably 0.5 to 10 mM, and morepreferably 1 to 3 mM. For liquid system analysis, the amount of mediatorwith respect to the total reagent solution ranges, e.g., from 0.1 to 10mM, preferably 0.1 to 1 mM, and more preferably 0.1 to 0.3 mM. Theseranges of optimum concentration depend on the type of mediator to beused.

EXAMPLES

Hereinafter, examples of the present invention will be described. Ineach of the examples, PQQ represents pyrroloquinoline quinone, and otherreagents are explained in detail in the following table. ReagentManufacturer Note (name, etc.) PQQGDH TOYOBO Co., Ltd PQQ-glucosedehyrogenase GOD Sigma Glucose oxidase type X-S PyruvateBoehringerMannheim oxidase Glucose Wako Pure Chemical D(+)−glucoseIndustries, Ltd. Pyruvic acid Wako Pure Chemical Lithium pyruvatemonohydrate Industries, Ltd.

Example 1

An aqueous solution of [Cu(bpy)₂]Cl₂.6H₂O (80 mM) was prepared by mixingCuCl₂ and 2,2′-bipyridyl at a molar ratio of 1:2 in a water bath atabout 80° C. HPC-M was dissolved in the aqueous solution of bipyridylcopper complex at 2 wt %, and then was heated to 50° C. and cooled to25° C. Further, GDH was dissolved in this aqueous solution at 50000U/ml, thus producing a reagent solution. Next, an asymmetrical porousfilm (“BTS-25”, manufactured by US Filter) in which pore sizes vary inthe thickness direction was impregnated from the surface that includessmaller pores with 2 wt % of an aqueous solution of inorganic gel(“Laponite XLG”, manufactured by Rockwood Additives Limited), and thenwas dried. Moreover, 2 μl of the reagent solution was applied to thesurface of the porous film that includes larger pores, and then wasair-dried to form a circular spot (light blue). This spot portion wascut and sandwiched between PET films with holes, so that an intendedtest piece for glucose analysis was obtained.

Serum having four different glucose concentrations (0 mg/ml, 2 mg/ml, 4mg/ml, and 6 mg/ml) was applied to the test piece, and color produced inthe test piece was measured after 5 seconds from the application byusing a reflectance measuring device (wavelength: 470 nm). The graph inFIG. 1 shows the results. As shown in FIG. 1, the test piece producedcolor in accordance with the glucose concentration within 5 seconds fromthe application. This color (reddish brown) corresponding to the glucoseconcentration also was observed visually. The time required forproducing the color was 2 to 3 seconds. The glucose-containing serum wasprepared in the following manner. Human blood plasma was glycolyzedcompletely, frozen, and melted to produce serum. Then, glucose was addedto the serum in different concentrations as described above.

Example 2

Copper (II) chloride (0.01 mol) was dissolved in hot water (30 ml), towhich 2,2′-bipyridyl (0.02 mol) was added and stirred. Then, thesolution was cooled to precipitate hexahydrate as crystals, thusproducing [Cu(bpy)₂]Cl₂.6H₂O. The following reaction reagent (1 ml)including this copper complex was placed in a microcell having anoptical path length of 10 mm, and the absorption spectrum at awavelength of 300 nm to 900 nm was measured with a spectrophotometer(“V-550”, manufactured by JASCO Corporation) and identified as a blank(oxidized). A 500 mM glucose aqueous solution (10 μl) was added to themicrocell while stirring, and the spectrum was measured immediately. Thegraph in FIG. 2 shows the results. As shown in FIG. 2, the coppercomplex was reduced by the enzyme reaction, and the color production wasobserved in a short wavelength region. Reaction reagent composition[Cu(bpy)₂]Cl₂ 0.4 mM PIPES (pH 7.0) 50 mM PQQGDH 200 U/ml

Example 3

Copper (II) chloride (0.01 mol) and 2,2′-bipyridyl (0.033 mol) wereadded to a small amount of water and heated until they were dissolvedcompletely. Then, the solution was cooled to precipitate[Cu(bpy)₃]Cl₂.6H₂O as crystals. The following reaction reagent (1 ml)including this copper complex was placed in a microcell having anoptical path length of 10 mm, and the absorption spectrum at awavelength of 300 nm to 900 nm was measured with a spectrophotometer(“V-550”, manufactured by JASCO Corporation) and identified as a blank(oxidized). A 500 mM glucose aqueous solution (10 μl) was added to themicrocell while stirring, and the spectrum was measured immediately. Thegraph in FIG. 3 shows the results. As shown in FIG. 3, the coppercomplex was reduced by the enzyme reaction, and the color production wasobserved in a short wavelength region. Reaction reagent composition[Cu(bpy)₃]Cl₂ 0.4 mM PIPES (pH 7.0) 50 mM PQQGDH 200 U/ml

Example 4

Copper (II) chloride (0.01 mol) was dissolved in hot water (10 ml),ethylenediamine (en, 0.01 mol) was dissolved in hot water (10 ml), and2,2′-bipyridyl (0.01 mol) was dissolved in hot ethanol (10 ml). Thethree solutions were mixed together to make a blue solution. Thissolution was cooled and concentrated, thus producing needle crystals of[Cu(en)(bpy)]Cl₂. The following reaction reagent (1 ml) including thiscopper complex was placed in a microcell having an optical path lengthof 10 mm, and the absorption spectrum at a wavelength of 300 nm to 900nm was measured with a spectrophotometer (“V-550”, manufactured by JASCOCorporation) and identified as a blank (oxidized). A 500 mM glucoseaqueous solution (10 μl) was added to the microcell while stirring, andthe spectrum was measured immediately. The graph in FIG. 4 shows theresults. As shown in FIG. 4, the copper complex was reduced by theenzyme reaction, and the color production was observed in a shortwavelength region. Reaction reagent [Cu(en)(bpy)]Cl₂ 0.4 mM PIPES (pH7.0) 50 mM PQQGDH 200 U/ml

Example 5

An aqueous solution of copper chloride was prepared by dissolving 511 mg(3.0 mmol, 1.0 eq.) of copper (II) chloride dihydrate in hot water (10mL). Further, 408 mg (6.0 mmol, 2.0 eq.) of imidazole was dissolved inwater (10 mL) and 69 mg (3.0 mmol, 1.0 eq.) of 2,2′-bipyridyl wasdissolved in ethanol (10 mL), and then the two solutions were mixedtogether. This mixed solution was added to the aqueous solution ofcopper chloride, thus producing a deep blue solution of[Cu(Him)₂(bpy)]Cl₂. The following reaction regent (1 ml) including thiscopper complex was placed in a microcell having an optical path lengthof 10 mm, and the absorption spectrum at a wavelength of 300 nm to 900nm was measured with a spectrophotometer (“V-550”, manufactured by JASCOCorporation) and identified as a blank (oxidized). A 500 mM glucoseaqueous solution (10 μl) was added to the microcell while stirring, andthe spectrum was measured immediately. The graph in FIG. 5 shows theresults. As shown in FIG. 5, the copper complex was reduced by theenzyme reaction, and the color production was observed in a shortwavelength region. Reaction reagent [Cu(Him)₂(bpy)]Cl₂ 1 mM PIPES (pH7.0) 50 mM PQQGDH 1000 U/ml

Example 6

Copper (II) chloride (0.01 mol) was dissolved in hot water (10 ml),L-arginine (0.01 mol) was dissolved in hot water (10 ml), and2,2′-bipyridyl (0.01 mol) was dissolved in hot ethanol (10 ml). Thethree solutions were mixed together to make a deep blue solution. Thissolution was cooled and concentrated, thus producing needle crystals of[Cu(L-Arg)(bpy)]Cl₂. The following reaction reagent (1 ml) includingthis copper complex was placed in a microcell having an optical pathlength of 10 mm, and the absorption spectrum at a wavelength of 300 nmto 900 nm was measured with a spectrophotometer (“V-550”, manufacturedby JASCO Corporation) and identified as a blank (oxidized). A 500 mMglucose aqueous solution (10 μl) was added to the microcell whilestirring, and the spectrum was measured immediately. The graph in FIG. 6shows the results. As shown in FIG. 6, the copper complex was reduced bythe enzyme reaction, and the color production was observed in a shortwavelength region. Reaction reagent [Cu(L-Arg)(bpy)]Cl₂ 1 mM PIPES (pH7.0) 50 mM PQQGDH 1000 U/ml

Example 7

An asymmetrical porous film (“BTS-25”, manufactured by US Filter) inwhich pore sizes vary in the thickness direction was impregnated fromthe surface that includes smaller pores with 2 wt % of an aqueoussolution of inorganic gel having the following composition, and then wasdried. Moreover, 2 μl of a reagent solution having the followingcomposition was applied to the surface of the porous film that includeslarger pores, and then was air-dried to form a circular spot (lightblue). This spot portion was cut and sandwiched between PET films withholes, so that an intended test piece for glucose analysis was obtained.Inorganic gel aqueous solution composition

Smectite (which is the same as that used in Example 1) CHAPS(Surface-active agent) 1.8 wt % Reagent solution composition 0.4 wt %[Cu(bpy)₂]Cl₂ 80 mM [OsCl(Him)(dmbpy)₂]Cl₃  3 mM PQQ-GDH 1000 U/ml BSA  5% CHAPS 0.4%

Serum having four different glucose concentrations (0 mg/ml, 2 mg/ml, 4mg/ml, and 6 mg/ml) was applied to the test piece, and color produced inthe test piece was measured after 5 seconds from the application byusing a reflectance measuring device (wavelength: 470 nm). The graph inFIG. 7 shows the results. As shown in FIG. 7, the test piece producedcolor in accordance with the glucose concentration immediately after theapplication. This color (reddish brown) corresponding to the glucoseconcentration also was observed visually. Moreover, the color productionoccurred in an instant. The glucose-containing serum was prepared in thefollowing manner. Human blood plasma was glycolyzed completely, frozen,and melted to produce serum. Then, glucose with different concentrationsas described above was added to the serum.

Example 8

A 500 mM glucose aqueous solution (10 μl) was added to a reagentsolution (100 μl) having the following composition. Then, a change incolor of the reagent solution from green (the original color) to reddishbrown was observed visually within one minute. The original color of thereagent solution was made by mixing the yellow of a coenzyme (FAD)included in GOD and the blue of copper. Reagent solution composition GOD(manufactured by Sigma, specific activity 209 U/mg) 50 mg/ml[Cu(bpy)₂]Cl₂ 40 mM PIPES (pH 7.0) 50 mM CHAPS 0.1 wt %

Example 9

A 500 mM glucose aqueous solution (10 μl) was added to a reagentsolution (100 μl) having the following composition. Then, a change incolor of the reagent solution from green (the original color) to reddishbrown was observed visually within 5 seconds. The original color of thereagent solution was made by mixing the yellow of a coenzyme (FAD)included in GOD and the blue of copper. Reagent solution composition GOD(manufactured by Sigma, specific activity 209 U/mg) 50 mg/ml[Cu(bpy)₂]Cl₂ 40 mM [OsCl(Him)(dmbpy)₂]Cl₂ 0.3 mM PIPES (pH 7.0) 50 mMCHAPS 0.1 wt %

Example 10

A copper complex was prepared by using various ligands. Copper (II)chloride and each of the following ligands were mixed at a molar ratioof 1:2, dissolved in purified water, and incubated for 10 minutes in awater bath at about 80° C. so that the ligands were coordinated to themetal. Thus, complex solutions were obtained. Ligand ManufacturerComplex 1,10-phenanthroline Wako Pure Chemical[Cu(1,10-phenanthroline)₂] Industries, Ltd. bathophenanthroline WakoPure Chemical [Cu(bathophenanthroline)₂] Industries, Ltd.bathophenanthroline Nacalai Tesque, Inc. [Cu(bathophenanthrolinesulfonic acid sulfonic acid)₂] disodium salt 2,2′-bipyridyl Wako PureChemical [Cu(2,2′-bipyridyl)₂] Industries, Ltd. TPTZ DOJINDO [Cu(TPTZ)₂]LABORATORIES cuproin Wako Pure Chemical [Cu(cuproin)₂] Industries, Ltd.

Example 11

A copper mixed ligand complex was prepared by using each of thefollowing ligands and the bipyridyl compounds. Copper, each of thefollowing ligands, and the bipyridyl compounds were mixed at a molarratio of 1:2:1, dissolved in purified water, and incubated for 10minutes in a water bath at about 80° C. so that the ligands and thebipyridyl compounds were coordinated to the metal. Thus, complexsolutions were obtained. Ligand Manufacturer Complex L-arginine NacalaiTesgue, Inc. [Cu(L-Arg)(bpy)] ethylenediamine Nacalai Tesgue, Inc.[Cu(en)(bpy)] imidazole Wako Pure Chemical [Cu(Him)(bpy)] Industries,Ltd.

Example 12

An iron complex was prepared by using various ligands. Iron (III) 5chloride and each of the following ligands were mixed at a molar ratioof 1:3, dissolved in purified water, and incubated for 10 minutes in awater bath at about 80° C. so that the ligands were coordinated to themetal. Thus, complex solutions were obtained. Ligand ManufacturerComplex 1,10-phenanthroline Wako Pure Chemical[Fe(1,10-phenanthroline)₃] Industries, Ltd. bathophenanthroline WakoPure Chemical [Fe(bathophenanthroline)₃] Industries, Ltd.bathophenanthroline Nacalai Tesque, Inc. [Fe(bathophenanthrolinesulfonic acid sulfonic acid)₃] disodium salt 2,2′-bipyridyl Wako PureChemical [Fe(2,2′-bipyridyl)₃] Industries, Ltd. 4,4′-diamino-2,2′-Arkray, Inc. [Fe(4,4′-diamino-2,2′- bipyridyl (DA-bpy) bipyridyl)₃] TPTZDOJINDO [Fe(TPTZ)₃] LABORATORIES PDTS DOJINDO [Fe(PDTS)₃] LABORATORIESnitroso-PSAP DOJINDO [Fe(nitroso-PSAP)₃] LABORATORIES nitroso-ESAPDOJINDO [Fe(nitroso-ESAP)₃] LABORATORIES 1-nitroso-2-naphthol KANTOKAGAKU [Fe(1-nitroso-2- naphthol)₃] 2-amino-4-thiazole Lancaster[Fe(2-amino-4-thiazole acetic acid acetic acid)₃] 1,2-naphthoquinone-Nacalai Tesque, Inc. [Fe(1,2-naphthoquinone-4- 4-sulfonic acid sulfonicacid)₃] nitro-PAPS DOJINDO [Fe(nitro-PAPS)₃] LABORATORIES

Example 13

Two types of ruthenium complexes were prepared in the following manner.

[Ru(NH₃)6]

A commercially available ruthenium complex (manufactured by Aldrich,Hexaammineruthenium (III) chloride) was dissolved in water to obtain acomplex solution of [Ru(NH₃)₆].

[Ru(4,4′-diamino-2,2′-bipyridyl)₃]

Ligand

First, 11.8 g (63.0 mmol) of 2,2′-bipyridyl-N,N′-dioxide (manufacturedby Aldrich) was dissolved slowly in 120 ml of concentrated sulfuric acidcooled in an ice bath, and the solution was heated to 100° C. Then, aconcentrated sulfuric acid solution (100 ml) containing 64.0 g (630mmol) of potassium nitrate was slowly added dropwise and stirred for 1hour while heating. After reaction, the solution was cooled to roomtemperature, poured into crushed ice, and filtered. Thus, a solid of4,4′-dinitro-2,2′-bipyridyl-N,N′-oxide was obtained. Next, 7.0 g (25 mM)of 4,4′-dinitro-2,2′-bipyridyl-N,N′-oxide and 6.0 g of 10% palladiumcarbon were suspended in ethanol (23 ml) under Ar. To this solution wasadded dropwise an ethanol solution (47 ml) containing 6.3 g (126 mmol)of hydrazine monohydrate, followed by refluxing for 8 hours. Thesolution was cooled and filtered. The filtrate was concentrated andpurified by silica gel column chromatography. Thus,4,4′-diamino-2,2′-bipyridyl was obtained.

Synthesis

Ethylene glycol (10 mL) was placed in a 50 mL two-neck flask, in whichDA-bpy (0.2 g) and RuCl₃ (0.1 g) were dissolved and stirredsuccessively. The solution was heated by a mantle heater whilevigorously stirring under N₂, followed by refluxing for about 4 hours.

Purification

After stirring and cooling under N₂, the solution was moved to a 100 mLround bottom flask and washed with acetone (5 mL)+diethyl ether (20 mL).This washing of the solution with acetone (5 mL)+diethyl ether (20 mL)was repeated until the solvent (ethylene glycol) was removedsufficiently. The target substance thus washed was dissolved in ethanoland precipitated by the addition of diethyl ether. The target substancewas filtered while washing with diethyl ether and dried under reducedpressure. Thus, a solid of [Ru(4,4′-diamino-2,2′-bipyridyl)₃] wasobtained. This solid was dissolved in water to obtain a complexsolution.

Example 14

Two types of osmium complexes were prepared in the following manner.

[OsCl(Him)(dmbpy)2]

Synthesis

First, 2.00 g (4.56 mmol) of (NH₄)₂[OsCl₆] (manufactured by Aldrich) and1.68 g (9.11 mmol) of 4,4′-dimethyl-2,2′-bipyridyl (dmbpy, manufacturedby Wako Pure Chemical Industries, Ltd.) were refluxed in ethylene glycol(60 ml) for 1 hour under N₂. After cooling to room temperature, 1Msodium dithionite solution (120 ml) was added for 30 minutes, followedby cooling in an ice bath for 30 minutes. The precipitates were filteredunder reduced pressure and sufficiently washed with water (500 to 1000ml). Further, the precipitates were washed with diethyl ether two times,and then dried under reduced pressure. Thus, 1.5 to 1.7 g of[OSCl₂(dmbpy)₂] was obtained. Next, 1.56 g (2.60 mmol) of[OsCl₂(dmbpy)₂] and 0.36 g (5.2 mmol) of imidazole (Him) were refluxedin a water/methanol mixed solvent (50 ml) under N₂ for 2 hours. Aftercooling to room temperature, a saturated NaCl solution (300 ml) wasadded. The precipitates were filtered under reduced pressure, washedwith a saturated NaCl solution, and dried under reduced pressure. Thus,[OsCl(Him)(dmbpy)₂]Cl₂ was obtained.

Purification

The [OsCl(Him)(dmbpy)₂]Cl₂ was dissolved in the smallest possible amountof acetonitrile/methanol (1:1 v/v) and purified by column chromatography(absorbent: activated alumina, developing solvent:acetonitrile/methanol). The solvent was evaporated, and the residue wasdissolved in a small amount of acetone and reprecipitated with diethylether. The precipitates were filtered and dried under reduced pressure,and then dissolved in water. Thus, a complex solution was obtained.

[Os(Him)₂(dmbpy)2]

Synthesis

First, 2.00 g (4.56 mmol) of (NH₄)₂[OsCl₆] and 1.68 g (9.11 mmol) ofdmbpy were refluxed in ethylene glycol (60 ml) under N₂ for 1 hour.After cooling to room temperature, 1M sodium dithionite solution (120ml) was added for 30 minutes, followed by cooling in an ice bath for 30minutes. The precipitates were filtered under reduced pressure andsufficiently washed with water (500 to 1000 ml). Further, theprecipitates were washed with diethyl ether two times, and then driedunder reduced pressure. Thus, 1.5 to 1.7 g of [OSCl₂(dmbpy)₂] wasobtained. Next, 1.56 g (2.60 mmol) of [OsCl₂(dmbpy)₂] and 0.36 g (5.2mmol) of imidazole (Him) were refluxed in a 1,2-ethanedithiol solvent(50 ml) under N₂ for 2 hours. After cooling to room temperature, asaturated NaCl solution (300 ml) was added. The precipitates werefiltered under reduced pressure, washed with a saturated NaCl solution,and dried under reduced pressure. Thus, [Os(Him)₂(dmbpy)₂]Cl₂ wasobtained.

Purification

The [Os(Him)₂(dmbpy)₂]Cl₂ was dissolved in the smallest possible amountof acetonitrile/methanol (1:1 v/v) and purified by column chromatography(absorbent: activated alumina, developing solvent:acetonitrile/methanol). The solvent was evaporated, and the residue wasdissolved in a small amount of acetone and reprecipitated with diethylether. The precipitates were filtered and dried under reduced pressure,and then dissolved in water. Thus, a complex solution was obtained.

Example 15

Reagent solutions were prepared by mixing a complex includingphenanthroline ligands in NN coordination form, an enzyme, and a buffersolution with the following compositions 1 to 6. The spectrum of each ofthe reagent solutions was measured and identified as a blank. Further,glucose equivalent in amount to the complex was added to each of thereagent solutions, and the spectrum was measured after the color change.FIGS. 8A to 8F show the results. The complexes prepared in the aboveexamples were used. Reagent solution composition 1 (FIG. 8A) PQQ-GDH 50U/mL [Cu(1,10-phenanthroline)₂] 1 mM PIPES pH 7 50 mM Triton X-100 0.5%Reagent solution composition 2 (FIG. 8B) PQQ-GDH 50 U/mL[Fe(1,10-phenanthroline)₃] 0.1 mM PIPES pH 7 50 mM Triton X-100 0.5%Reagent solution composition 3 (FIG. 8C) PQQ-GDH 50 U/mL[Cu(bathophenanthroline)₂] 1 mM PIPES pH 7 50 mM Triton X-100 0.5%(bathophenanthroline = 4,7-diphenyl phenanthroline) Reagent solutioncomposition 4 (FIG. 8D) PQQ-GDH 50 U/mL [Fe(bathophenanthroline)₃] 1 mMPIPES pH 7 50 mM Triton X-100 0.5% (bathophenanthroline = 4,7-diphenylphenanthroline) Reagent solution composition 5 (FIG. 8E) PQQ-GDH 50 U/mL[Cu(bathophenanthroline sulfonic acid)₂] 1 mM PIPES pH 7 50 mM TritonX-100 0.5% Reagent solution composition (FIG. 8F) PQQ-GDH 50 U/mL[Fe(bathophenanthroline sulfonic acid)₃] 0.1 mM PIPES pH 7 50 mM TritonX-100 0.5%

Example 16

Reagent solutions were prepared by mixing a complex including bipyridylligands in NN coordination form, an enzyme, and a buffer solution withthe following compositions 1 to 4. The spectrum of each of the reagentsolutions was measured and identified as a blank. Further, glucoseequivalent in amount to the complex was added to each of the reagentsolutions, and the spectrum was measured after the color change. FIGS.9A to 9D show the results. The complexes prepared in the above exampleswere used. Reagent solution composition 1 (FIG. 9A) PQQ-GDH 50 U/mL[Cu(2,2′-bipyridyl)₂] 1 mM PIPES pH 7 50 mM Triton X-100 0.5% Reagentsolution composition 2 (FIG. 9B) PQQ-GDH 50 U/mL [Fe(2,2′-bipyridyl)₃] 1mM PIPES pH 7 50 mM Triton X-100 0.5% Reagent solution composition 3(FIG. 9C) PQQ-GDH 50 U/mL [Fe(4,4′-diamino-2,2′-bipyridyl)₃] 0.1 mMPIPES pH 7 50 mM Triton X-100 0.5% Reagent solution composition 4 (FIG.9D) PQQ-GDH 50 U/mL [Ru(4,4′-diamino-2,2′-bipyridyl)₃] 10 mM PIPES pH 750 mM Triton X-100 0.5%

Example 17

Reagent solutions were prepared by mixing a complex including triazineligands in NN coordination form, an enzyme, and a buffer solution withthe following compositions 1 to 3. The spectrum of each of the reagentsolutions was measured and identified as a blank. Further, glucoseequivalent in amount to the complex was added to each of the reagentsolutions, and the spectrum was measured after the color change. FIGS.10A to 10C show the results. The complexes prepared in the aboveexamples were used. Reagent solution composition 1 (FIG. 10A) PQQ-GDH 50U/mL [Cu(TPTZ)₂] 1 mM PIPES pH 7 50 mM Triton X-100 0.5% (TPTZ =2,4,6-tripyridyl-s-triazine) Reagent solution composition 2 (FIG. 10B)PQQ-GDH 50 U/mL [Fe(TPTZ)₃] 0.1 mM PIPES pH 7 50 mM Triton X-100 0.5%(TPTZ = 2,4,6-tripyridyl-s-triazine) Reagent solution composition 3(FIG. 10C) PQQ-GDH 50 U/mL [Fe(PDTS)₃] 1 mM PIPES pH 7 50 mM TritonX-100 0.5% (PDTS = 3-(2-pyridyl)-5,6-bis(4-sulfophenyL)-1,2,4-triazine)

Example 18

A reagent solution was prepared by mixing a complex includingbiquinoline ligands in NN coordination form, an enzyme, and a buffersolution with the following composition. The spectrum of the reagentsolution was measured and identified as a blank. Further, glucoseequivalent in amount to the complex was added to the reagent solution,and the spectrum was measured after the color change. FIG. 11 shows theresults. The complex prepared in the above examples was used.

Reagent Solution Composition PQQ-GDH 50 U/mL [Cu(cuproin)₂] 1 mM PIPESpH 7 50 mM Triton X-100 0.5%(cuproin = 2,2′-biquinoline)

Example 19

A reagent solution was prepared by mixing a complex including pyridylazoligands in NN coordination form, an enzyme, and a buffer solution withthe following composition. The spectrum of the reagent solution wasmeasured and identified as a blank. Further, glucose equivalent inamount to the complex was added to the reagent solution, and thespectrum was measured after the color change. FIG. 12 shows the results.The complex prepared in the above examples was used. Reagent solutioncomposition PQQ-GDH 50 U/mL [Fe(nitro-PAPS)₃] 0.02 mM PIPES pH 7 50 mMTriton X-100 0.5%(nitro-PAPS =2-(5-nitro-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)aminophenol])

Example 20

Reagent solutions were prepared by mixing a complex including ligands inNO coordination form, an enzyme, and a buffer solution with thefollowing compositions 1 to 3. The spectrum of each of the reagentsolutions was measured and identified as a blank. Further, glucoseequivalent in amount to the complex was added to each of the reagentsolutions, and the spectrum was measured after the color change. FIGS.13A to 13C show the results. The complexes prepared in the aboveexamples were used. Reagent solution composition 1 (FIG. 13A) PQQ-GDH 50U/mL [Fe(nitroso-PSAP)₃] 0.05 mM PIPES pH 7 50 mM Triton X-100 0.5%(nitroso-PSAP = 2- nitroso-5-[N-n-propyl-N-(3-sulfopropyl)aminophenol])Reagent solution composition 2 (FIG. 13B) PQQ-GDH 50 U/mL[Fe(nitroso-ESAP)₃] 0.1 mM PIPES pH 7 50 mM Triton X-100 0.5%(nitroso-ESAP = 2-nitroso-5-[N-ethyl-N- (3-sulfopropyl)aminophenol])Reagent solution composition 3 (FIG. 13C) PQQ-GDH 50 U/mL[Fe(1-nitroso-2-naphthol)₃] 0.1 mM PIPES pH 7 50 mM Triton X-100 0.5%

Example 21

A reagent solution was prepared by mixing a complex including ligands inNS coordination form, an enzyme, and a buffer solution with thefollowing composition. The spectrum of the reagent solution was measuredand identified as a blank. Further, glucose equivalent in amount to thecomplex was added to the reagent solution, and the spectrum was measuredafter the color change. FIG. 14 shows the results. The complex preparedin the above examples was used. Reagent solution composition PQQ-GDH 50U/mL [Fe(2-amino-4-thiazoleacetic acid)₃] 1 mM PIPES pH 7 50 mM TritonX-100 0.5%

Example 22

A reagent solution was prepared by mixing a complex including ligands inOO coordination form, an enzyme, and a buffer solution with thefollowing composition. The spectrum of the reagent solution was measuredand identified as a blank. Further, glucose equivalent in amount to thecomplex was added to the reagent solution, and the spectrum was measuredafter the color change. FIG. 15 shows the results. The complex preparedin the above examples was used. Reagent solution composition PQQ-GDH 50U/mL [Fe(1,2-naphthoquinone-4-sulfonic acid)₃] 1 mM PIPES pH 7 50 mMTriton X-100 0.5%

Example 23

Reagent solutions were prepared by mixing a mixed ligand complex, anenzyme, and a buffer solution with the following compositions 1 to 5.The spectrum of each of the reagent solutions was measured andidentified as a blank. Further, glucose equivalent in amount to thecomplex was added to each of the reagent solutions, and the spectrum wasmeasured after the color change. FIGS. 16A to 16E show the results. Thecomplexes prepared in the above examples were used. Reagent solutioncomposition 1 (FIG. 16A) PQQ-GDH 50 U/mL [OsCl(Him)(dmbpy)₂] 0.1 mMPIPES pH 7 50 mM Triton X-100 0.5% (Him = imidazole) (dmbpy =4,4′-dimethyl-2,2′-bipyridyl) Reagent solution composition 2 (FIG. 16B)PQQ-GDH 50 U/mL [Os(Him)₂(dmbpy)₂] 0.1 mM PIPES pH 7 50 mM Triton X-1000.5% Reagent solution composition 3 (FIG. 16C) PQQ-GDH 50 U/mL[Cu(L-Arg)₂(bpy)] 1 mM PIPES pH 7 50 mM Triton X-100 0.5% (L-Arg =L-arginine) (bpy = 2,2′-bipyridyl) Reagent solution composition 4 (FIG.16D) PQQ-GDH 50 U/mL [Cu(en)₂(bpy)] 1 mM PIPES pH 7 50 mM Triton X-1000.5% (en = ethylenediamine) (bpy = 2,2′-bipyridyl) Reagent solutioncomposition 5 (FIG. 16E) PQQ-GDH 50 U/mL [Cu(Him)₂(bpy)] 1 mM PIPES pH 750 mM Triton X-100 0.5%

Example 24

Reagent solutions were prepared by mixing a complex, an enzyme (glucoseoxidase (GOD) or pyruvate oxidase), and a buffer solution with thefollowing compositions 1 and 2. The spectrum of each of the reagentsolutions was measured and identified as a blank. Further, glucose orpyruvic acid equivalent in amount to the complex was added to each ofthe reagent solutions, and the spectrum was measured after the colorchange. FIGS. 17A and 17B show the results. The complexes prepared inthe above examples were used. Reagent solution composition 1 (FIG. 17A)GOD 100 U/mL [Cu(2,2′-bipyridyl)₂] 1 mM PIPES pH 7 50 mM Triton X-1000.5% Reagent solution composition 2 (FIG. 17B) pyruvate oxidase 100 U/mL[OsCl(Him)(dmbpy)₂] 0.1 mM PIPES pH 7 50 mM Triton X-100 0.5% (Him =imidazole) (dmbpy = 4,4′-dimethyl-2,2′-bipyridyl)

Example 25

A reagent solution was prepared with the following composition.Moreover, the following enzyme solutions 1, 2, and 3 were prepared.Then, 10 μL of glucose aqueous solution (with concentrations of 0, 10,20, and 30 mM and final concentrations of 0, 0.1, 0.2, and 0.3 mM,respectively) and 500 μL of the reagent solution were mixed in adispocell having an optical path length of 10 mm, to which 500 μL ofeach of the enzyme solutions was added to cause a reaction between thesolutions. The absorbance change was measured for 50 seconds with aspectrophotometer (wavelength: 600 nm). FIGS. 18A, 18B, and 18C show theresults. As shown in FIGS. 18A, 18B, and 18C, the reaction rateincreased with the amount of enzyme. When the enzyme activity was 1000U/mL, the reaction came to an end in about 5 seconds. It is possible toquantify the glucose concentration by sampling signals near 5 seconds,at which the reaction reaches the end. The slope of the graphs from thebeginning to the end of the reaction also can be used to quantify theglucose concentration. Reagent solution composition Cu(PDTS)₂ 1 mM PIPESpH 7 50 mM Triton X-100 0.5% Enzyme solution 1 (FIG. 18A) PQQ-GDH 111U/mL Enzyme solution 2 (FIG. 18B) PQQ-GDH 333 U/mL Enzyme solution 3(FIG. 18C) PQQ-GDH 1000 U/mL

Industrial Applicability

As described above, a calorimetric method of the present invention canperform simple and reliable analysis in a short time.

1. A colorimetric method comprising: allowing an oxidoreductase to acton an analyte and a color-changeable substance that changes color bytransferring an electron; and performing a qualitative or quantitativeanalysis of the analyte by measuring color produced in thecolor-changeable substance due to electron transfer from the analyte tothe color-changeable substance.
 2. The calorimetric method according toclaim 1, wherein the color-changeable substance that changes color bytransferring an electron is a transition metal complex.
 3. Thecolorimetric method according to claim 2, wherein the transition metalcomplex is at least one complex selected from the group consisting of acopper complex, an iron complex, a ruthenium complex, and an osmiumcomplex.
 4. The colorimetric method according to claim 3, wherein acoordinating atom of a ligand in the transition metal complex is atleast one selected from the group consisting of nitrogen, oxygen, andsulfur.
 5. The colorimetric method according to claim 4, wherein theligand in the transition metal complex is selected from the groupconsisting of ammonia, a bipyridyl compound, an imidazole compound, aphenanthroline compound, an ethylenediamine compound, amino acids, atriazine compound, a biquinoline compound, a pyridylazo compound, anitroso compound, an oxine compound, a benzothiazole compound, anacetylacetone compound, an anthraquinone compound, a xanthene compound,oxalic acid, and a derivative of each of the compounds.
 6. Thecolorimetric method according to claim 5, wherein at least one hydrogenatom that occupies a position other than a coordination position of theligand is replaced by a substituent.
 7. The colorimetric methodaccording to claim 6, wherein the substituent is at least one selectedfrom the group consisting of an alkyl group, an aryl group, an allylgroup, a phenyl group, a hydroxyl group, an alkoxy group, a carboxylgroup, a carbonyl group, a sulfone group, a sulfonyl group, a nitrogroup, a nitroso group, a primary amine, a secondary amine, a tertiaryamine, an amino group, an acyl group, an amido group, and a halogengroup.
 8. The colorimetric method according to claim 2, wherein thetransition metal complex includes two or more types of ligands.
 9. Thecolorimetric method according to claim 1, wherein the oxidoreductase isa dehydrogenase or an oxidase.
 10. The calorimetric method according toclaim 1, wherein the analyte is glucose, creatine, creatinine,cholesterol, uric acid, pyruvic acid, or lactic acid, and theoxidoreductase is a dehydrogenase or an oxidase that corresponds to therespective analyte.
 11. A reagent used for the colorimetric methodaccording to claim 1 comprising: an oxidoreductase; and acolor-changeable substance that changes color by transferring anelectron.
 12. The reagent according to claim 11, wherein thecolor-changeable substance that changes color by transferring anelectron is a transition metal complex.
 13. The reagent according toclaim 12, wherein the transition metal complex is at least one complexselected from the group consisting of a copper complex, an iron complex,a ruthenium complex, and an osmium complex.
 14. The reagent according toclaim 12, wherein a coordinating atom of a ligand in the transitionmetal complex is at least one selected from the group consisting ofnitrogen, oxygen, and sulfur.
 15. The reagent according to claim 14,wherein the ligand in the transition metal complex is selected from thegroup consisting of ammonia, a bipyridyl compound, an imidazolecompound, a phenanthroline compound, an ethylenediamine compound, aminoacids, a triazine compound, a biquinoline compound, a pyridylazocompound, a nitroso compound, an oxine compound, a benzothiazolecompound, an acetylacetone compound, an anthraquinone compound, axanthene compound, oxalic acid, and a derivative of each of thecompounds.
 16. The reagent according to claim 15, wherein at least onehydrogen atom that occupies a position other than a coordinationposition of the ligand is replaced by a substituent.
 17. The reagentaccording to claim 16, wherein the substituent is at least one selectedfrom the group consisting of an alkyl group, an aryl group, an allylgroup, a phenyl group, a hydroxyl group, an alkoxy group, a carboxylgroup, a carbonyl group, a sulfone group, a sulfonyl group, a nitrogroup, a nitroso group, a primary amine, a secondary amine, a tertiaryamine, an amino group, an acyl group, an amido group, and a halogengroup.
 18. The reagent according to claim 12, wherein the transitionmetal complex includes two or more types of ligands.
 19. The reagentaccording to claim 11, wherein the oxidoreductase is a dehydrogenase oran oxidase.
 20. The reagent according to claim 11, wherein the analyteis glucose, cholesterol, uric acid, pyruvic acid, creatine, creatinine,or lactic acid, and the oxidoreductase is a dehydrogenase or an oxidasethat corresponds to the respective analyte.
 21. A test piece comprisingthe reagent according to claim
 11. 22. The test piece according to claim21, further comprising an inorganic gel.